"Who" can help you easily detect cell viability
Cell Counting Kit is abbreviated as CCK-8 kit, which is based on WST-8 (chemical name: 2-(2-methoxy-4-nitrobenzene)-3-(4-nitrophenyl)-5-( 2,4-Disulfobenzene)-2H-tetrazole monosodium salt) is a rapid and highly sensitive detection kit widely used in cell proliferation and cytotoxicity. WST-8 can be reduced by dehydrogenase in mitochondria to a highly water-soluble yellow formazan product in the presence of electronic coupling reagents. The intensity of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. Use a microplate reader to measure the OD value at 450nm wavelength, which indirectly reflects the number of living cells. CCK-8 has been widely used in high-throughput drug screening, cell proliferation determination, cytotoxicity determination, tumor drug susceptibility test, and activity detection of biological factors.
Basic operation method
1. Use a 96-well plate, add 100-200 μL of medium to each well, containing 1000-2000 cells. The number of cells depends on the size of the cells, the speed of cell proliferation and other factors. The number of inoculated holes is set according to the specific experimental requirements.
2. In order to reduce the adverse effects of the edge effect on the cells in the wells, fill up the wells around the experimental wells with 250 μL PBS and place them in the incubator for the required number of days.
3. Before the experiment, take CCK-8 to room temperature to dissolve in equilibrium, and then prepare an appropriate amount of complete medium containing CCK-8 according to the ratio of CCK-8 solution: medium = 1:10. This process needs to be protected from light, and the prepared medium also needs to be placed and added in the dark.
4. Discard the supernatant of each well in the 96-well plate, add PBS to the control group, add 100-200 μL of complete medium containing CCK-8 to the experimental group in the dark, and place it in an incubator for incubation.
Note: In order to ensure the same ratio of CCK-8 solution and medium in each well, mix the two first and then uniformly add them. If the error between the wells is not considered, or for the convenience of experimental operation (such as viability determination of paraffin-enclosed cells), calculate the amount of CCK-8 to be added according to the volume of the medium in the well, and directly absorb CCK-8 The solution is added to the well.
5. After incubating for 2 hours (the length of time depends on the cell type and cell density, etc.), there is no need for any treatment, and the 96-well plate is processed as soon as possible under the dark condition.
6. Place the 96-well plate in the microplate reader to determine the OD value. The wavelength is set to 450nm, after obtaining the data results, export the Excel sheet for subsequent analysis.
7. Remove the mixed solution in the 96-well plate, rinse it with clean water, and discard it.
Operation methods for different application scenarios
1. Cell growth curve: The number of days for growth curve determination is 1-7 days. The number of 96-well plates is prepared according to the number of days, and one plate is consumed every day. The number of multiple wells of each group on each plate, the number of cells to be inoculated, the medium and volume used, the amount of CCK-8 solution added and the duration of action must be consistent.
2. The promotion/inhibition effect of drugs, factors, and culture systems on cell growth: In order to ensure the consistency of cells in each group of each plate, it is best to first uniformly inoculate cells and then change to a special culture system the next day. When inoculating, prepare an additional control plate that has undergone the same treatment for the determination of the OD value of the next day to determine the initial data volume. After replacing the special culture system, the sample plate can be selected for CCK-8 detection at a specific time according to the experimental plan, and combined with the initial OD value data for analysis.
Traditional cell viability detection method: MTT method
MTT method, also known as MTT colorimetric method, is a method for detecting cell survival and growth. The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT to water-insoluble blue-purple crystal formazan (Formazan) and deposit it in the cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at 540 or 720nm with an enzyme-linked immunoassay, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells. This method has been widely used in the activity detection of some biologically active factors, large-scale anti-tumor drug screening, cytotoxicity test, and tumor radiosensitivity determination.
Operational difference between MTT method and CCK-8 method
1. Due to the water solubility of CCK-8, it does not need to be dissolved in DMSO after the effect is completed, and can be directly measured on the machine; while for the MTT method, the supernatant needs to be removed, and 150μL of DMSO is added to each well to dissolve the insoluble blue-purple crystal formazan at the bottom of the well.
2. CCK-8 generally has a shorter duration of action, usually 2-3h, and MTT is 4h
3. The wavelength used by the microplate reader is different. CCK-8 measures the OD value at 450nm wavelength. Due to the limitation of the instrument parameters, the OD value is usually measured at 490nm wavelength in actual operation.
4. CCK-8 is less toxic to cells and relatively safer
